This invention relates to a method and apparatus for performing capillary electrophoresis. More particularly, this invention relates to an improved method and apparatus for capillary electrophoresis that permits concentration of solutes from a solution prior to separating the solutes by capillary electrophoresis.
Capillary electrophoresis (CE) is an efficient analytical separation technique for analysis of minute amounts of sample. CE separations are performed in a narrow diameter capillary tube, which is filled with an electrically conductive medium termed the "carrier electrolyte." An electric field is applied between the two ends of the capillary tube, and species in the sample move from one electrode toward the other electrode at a rate which is dependent on the electrophoretic mobility of each species as well as on the rate of fluid movement in the tube. CE may be performed using gels or liquids, such as buffers, in the capillary. In one liquid mode, known as free zone electrophoresis, separations are based on differences in the free solution mobility of sample species. In another liquid made, micelles are used to effect separations based on differences in hydrophobicity. This is known as Micellar Electrokinetic Capillary Chromatography (MECC).
CE is advantageous for several reasons. These include fast separation speed, high resolution and small sample size. For example, separation speeds using CE can be 10 to 20 times faster than conventional gel electrophoresis, and no post-run staining is necessary. In part, high resolution can be obtained through the use of high voltages because of the rapid dissipation of heat by the capillary. Further, band broadening is minimized due to the narrow capillary inner diameter. In free-zone electrophoresis, the phenomenon of electroosmosis, or electroosmotic flow (EOF) occurs. This is a bulk flow of liquid which affects all of the sample molecules regardless of charge. Under certain conditions EOF can contribute to improved resolution and separation speed in free-zone CE.
In order to achieve the high resolution that CE is capable of, it is necessary that the sample be confined to a narrow starting zone when the electrophoretic process begins. This limits the volume of sample that can be introduced into the capillary to a very small fraction of the total capillary volume. Further, the lowest concentration of material that can be detected in CE by ultraviolet and visible absorbance detection is severely limited by the small inner diameter of the capillary, since absorbance detection is generally carried out with a beam of radiation that is transverse to the axis of the capillary. These facts, taken together, mean that CE has not been able to analyze samples at concentrations as low as those that can be analyzed by other techniques such as liquid chromatography. One approach to overcome this limitation has been described by several investigators. This approach involves dissolving the sample in a buffer or electrolyte having an ionic strength substantially lower than the carrier electrolyte used to carry out the electrophoretic separation. The sample so dissolved is introduced into the capillary in the normal fashion although a larger sample volume is now permitted. When the electric field is applied to begin the electrophoretic separation, the field strength in the sample zone will be higher than in the surrounding carrier electrolyte because the ionic strength and hence the conductivity is lower in the sample zone. As a result of the higher field strength in the sample zone, the ions in the sample will migrate at a higher speed than ions in the carrier electrolyte. Thus the sample ions will tend to pile up at the interface of the low conductivity sample zone and the carrier electrolyte because they will slow down once they enter the carrier electrolyte. This process effectively produces a narrow sample zone and has allowed an improvement of perhaps a factor of five in the lowest concentration of sample that can be analyzed.
A technique for preconcentrating a sample prior to electrophoretic separation was reported by Aebersold and Morrison at the High Performance Capillary Electrophoresis Symposium in January 1990. The inner surface of a short piece of a capillary tubing was coated with an adsorptive coating. This tube was then butted up against the end of an uncoated capillary tube. This arrangement permitted a large volume of sample (which would normally have caused severely broadened peaks) to be injected into the coated length of tube and then into the uncoated tube. The sample molecules adsorbed on the coating while the solvent passed through the tube and was washed out of the capillary tube with electrolyte. A small volume of organic solvent was then introduced into the butted capillary tubes. This solvent caused the sample molecules to desorb from the coating so that they entered the uncoated tube in concentrated form. Because the organic solvent had lower conductivity than the carrier electrolyte, the previously mentioned zone narrowing then occured. With both ends of the capillary tube in electrolyte, capillary electrophoretic separation was then performed in normal fashion. It was reported that this preconcentration step allows the use of CE with samples 5 to 10 times lower in concentration than was previously possible with CE.
Packed capillary columns have been used in liquid chromatography to effect sample separations. Such columns have been constructed in various ways. One way of making such a column is disclosed by Jorgenson et al in Journal of Chromatography, Vol. 218 (1981) pgs. 209-216. The column is formed by filling one end of a capillary tube with a particulate packing. The filled end then is heated in a flame to sinter the packing to form a porous frit. A slurry of chromatographic separation particles is introduced into the end of the capillary opposite to the fritted end until the capillary is filled with these particles. A second frit then is formed in the open end of the capillary in the same manner as was the first frit.
As disclosed in U.S. Pat. No. 4,793,920 to Cortes et al, a packed silica capillary chromatography column is produced by casting a plug of ceramic material in one end of the capillary to form a porous plug which is fused such as with heat. The chromatographic packing then is introduced into the remainder of the tube. U.S. Pat. No. 4,483,773 to Yang also discloses a means for forming a plug and chromatographic packing within a capillary chromatographic column.
It would be desirable to provide a means whereby dilute sample solution can be analyzed by CE at concentrations far lower than can be analyzed by presently available CE processes.